Design,Synthesis And Imaging Of Fluorescent Probes For Tumor Diagnosis In Vitro

With the improvement of clinical requirements for disease detection,traditional detection mothods are gradually unable to satisfy the demand.Especially for tumors,it requires the diagnosis with higher sensitivity and accuracy because these can significantly improve the cure rate of tumors and increase the survival rate of cancer patients.Among the various detection methods,fluorescent probe is powerful tool for analyzing sensing and optical imaging,which resultes from that it can directly visualize the biological analytes at the molecular level and provide useful information for the complex biological structures and processes.Various biomarkers in the cell,such as DNA,RNA and biological enzymes,can be detected by fluorescent probe.Thus,fluorescent probes have great potential for visual detection of tumor cells.However,conventional fluorophores are mostly insoluble in water,and fluorescence will be quenched due to aggregation of fluorophores in aqueous solution.This phenomenon is known as aggregation-caused quenching?ACQ?,so that the fluorescent probes designed based on ACQ fluorophore can only emit light in a low concentration aqueous solution,and are very susceptible to interference of false-positive signal.These defects limit the application in the hydrophilic environment of organisms and reduce the accuracy of the analysis results.Therefore,how to use fluorescent probes to accurately acquire important information of living organisms and diagnose tumor at an early stage is still a challenge so far.At present,the principle of fluorescence resonance energy transfer?FRET?is widely used in the design of various fluorescent probes.The background signal and false-positive signal of ACQ fluorescent probes could be reduced to some extent if they were designed by using FRET principle.Unfortunately,the defects causing by ACQ fluorophore cannot be fundamentally eliminated.In 2001,Tang and coworkers first discovered and proposed the aggregation-induced emission?AIE?phenomenon opposite to the ACQ phenomenon.In contrast to ACQ fluorophore,AIE fluorophore faintly illuminate or illuminate in dilute solutions and bright fluorescence is emitted when they are in a state of aggregation or solid state.The characteristic of AIE fluorophore can fundamentally avoid these drawbacks of ACQ fluorophore and provide unparalleled advantages in bioassay.Based on AIE and FRET principle,the paper provides several fluorescent probe detection strategies for improving the accuracy of detection:?1?a strategy to improve the contrast of the imaging;?2?a fluorescent cocktail nanoparticle for dual tumor markers collaborative imaging;?3?a probe to distinguish specific signal and false-positive signal by different fluorescence signals.First,an AIE fluorescent probe for in situ detection of alkaline phosphatase?ALP?activity in tumor cells was designed by modifying a phosphate group on the AIEgen,which could increase the contrast of the imaging and make the detection result more accurate.However,since the detection of dual tumor markers can significantly improve the accuracy of detection results compared with single tumor marker detection,we designed a fluorescent cocktail nanoparticle to achieve the synergy between intracellular detection of mRNA and H2O2by using FRET principle.The nanoparticle could simultaneously separate tumors,inflammation and normal cells,which significantly improved the accuracy of tumor cell detection.Finally,in order to completely avoid the false-positive signal of nucleic acid probes,an AIE-molecular beacon?AIE-MB?was designed on the basis of a novel AIEgen.AIE-MB could simultaneously detect the target mRNA in tumor cells and the false-positive signal in normal cells,which significantly improved the accuracy of the tumor detection result.Main contents are as follows.1.In Situ Localization of Alkaline Phosphatase Activity in Tumor Cells by an Aggregation-Induced Emission Fluorophore-Based ProbesConventional enzyme-responsive fluorescent probes are difficult to detect their targeted enzymes in situ in cells and low detection accuracy because their fluorescence can spread to other location from the reaction site.In order to solve the problem,we designed and synthesized an enzyme-responsive,water-soluble fluorescent probe with AIE characteristics.The AIE fluorophore?TPEQH?was utilized to design the enzyme-responsive,fluorescent probe?TPEQHA?by introducing phosphate group,which could be specifically degraded by ALP.In tumor cells,the phosphate on TPEQHA could be specifically degraded due to the highly expressed ALP and produced water-insoluble TPEQH.TPEQH could precipitated and release fluorescence in situ,thereby successfully detecting ALP by the signal.Furthermore,the expression level of ALP could be determined by the fluorescence intensity of TPEQH,and the detection result were more accurate.This demonstrated potential application value in both tumor diagnosis and biomedical research.2.A Fluorescent Cocktail Strategy for Differentiating Tumor,Inflammation and Normal Cells by Detecting mRNA and H₂O₂A fluorescent cocktail nanoparticle capable of simultaneously detecting intracellular mRNA and H2O2 was designed to differentiate tumors from nontumor cells.To detect targeted mRNA in living cells,a DNA probe was generated using FRET principle.A pH-responsive amphiphilic polymer was synthesized to realize the transportation of the DNA probe.In addition,the polymer was conjugated with a coumarin-boronic acid ester?Cou-BE?H2O2 probe.According to the change in the fluorescence of Cou-BE,tumor and inflammatory cells could be distinguished from normal cells owing to their high concentration of H2O2.Due to the different concentrations of tumor-related mRNA in tumor and nontumor cells,the fluorescence intensity of the DNA probe-loaded nanoparticles inside tumor cells was different from that inside inflammatory cells.Therefore,our fluorescent cocktail strategy could discriminate simultaneously tumor,inflammation and normal cells through the cooperative detection of intracellular mRNA and H2O2,which improved the accuracy of the tumor detection result and demonstrated potential application value in biomedical research and clinical diagnosis.3.Aggregation-Induced Emission Fluorophore-Based Molecular Beacon for Differentiating Tumor and Normal Cells by Detecting the Specific and False-Positive SignalsMajority of developed nucleic acid-based fluorescent probes for imaging intracellular mRNA were only designed to detect the target mRNA but could not avoid the interference arising from nuclease or other biological matrices,which results in inevitable false-positive signals.To overcome this dilemma,a new AIE fluorophore and FRET principle were used to establish a novel AIE fluorophore-based molecular beacon?AIE-MB?.The new AIE fluorophore?tetraphenylethylene-quinoxaline,TPEQ?was designed by incorporating an ACQ fluorophore?quinoxalinone?with one typical AIE active luminogen?tetraphenylethene,TPE?.On this basis,the AIE-MB was designed by labeling two fluorophores:the TPEQ?acceptor?conjugate and an ACQ fluorophore conjugate,coumarin?AMCA,donor?.Based on the two fluorophores,the AIE-MB could exhibit three states:weak fluorescence at primary stage,blue fluorescence?specific signal?generated by pairing with target mRNA in tumor cells,both green and blue fluorescence?false-positive signal?due to the endogenous degradation in normal cells.Obviously,the specific imaging for target mRNA in tumor cells and the false-positive signal resulting from endogenous degradation in normal cells could be intuitively distinguished through the different fluorescence emission.As a result,in contrast to traditional nucleic acid-based fluorescent probes,the AIE-MB could improve the accuracy of the tumor detection result by efficiently detecting specific and false-positive signals,which showed potential application value in both tumor diagnosis and biomedical research.